Bacterial infestation either in the medical field or in commercial non-medical field is unwelcome. To effectively eliminate unwanted bacterial growth, researchers have looked into the basic building blocks of bacteria, such as the cell wall of bacteria. One of the major components of a bacterial cell wall is the peptidoglycans, whose subunits are known as muramyl peptides. Peptidoglycans, which are formed from subunits of muramyl peptides, undergo cycles of assembly and disassembly required for cell wall remodeling during cell growth and division. Muramyl peptides are generated during the disassembly and recycled to construct new peptidoglycans during cell growth and septation. Accordingly, muramyl peptides are constantly released from many bacterial species during proliferation. They are also generated and released when bacterial cells are lysed by phages, antibiotics and host phagocytes.
Many muramyl peptides are potent signaling molecules and have been shown to strongly influence multiple physiological processes in the human host. Biological activities that have been linked to muramyl peptides include adjuvanticity, somnogenicity, pyrogenicity, and toxicity to ciliated epithelial cells. Muramyl peptides have also been associated with a range of human diseases such as sepsis, Crohn's disease, rheumatoid arthritis, asthma, allergy, atopic disorders, multiple sclerosis, pertussis, and gonorrhea. Different types of muramyl peptides carrying different side chains on the muramic acid residues and different amino acid residues in the peptides have distinct activities.
Muramyl peptides impact the human physiology by binding to specific peptidoglycan recognition proteins (PGRPs). Humans have four PGRPs, which are able to directly bind to both Gram-positive and Gram-negative peptidoglycan, and two intracellular peptidoglycan sensors, Nod1 and Nod2 belonging to a large family of pattern recognition receptors that recognize conserved microbe- or pathogen-associated molecular patterns. Nod2 recognizes the muramyl dipeptide N-acetylmuramyl-L-alanyl-D-glutamine, while Nod1 recognize the D-γ-glutamyl-mDAP motif in the peptide. Both Nod1. and Nod2 trigger and regulate the host immune response by activating the transcription factor NT-κB, which in turn switches on the production of proinflammatory cytokines and chemokines and expression of other defense genes. Nod1, Nod2 and NF-κB have been implicated in numerous human diseases.
The human microbiota, which contains 10 to 100 trillion non-pathogenic bacterial cells, constantly produces and secretes large quantities of muramyl peptides. A fraction of these molecules can enter the host circulation system via various mechanisms during the homeostatic state of the host as well as under numerous pathophysiological conditions. In addition to the many established links to human diseases as described above, strong evidence is rapidly accumulating that muramyl peptides released by commensal bacteria play key roles in the development, maintenance and modulation of major human systems such as the immune system, the gastrointestinal barrier, and the brain. Thus, the levels and types of muramyl peptides in the human body are thought to contribute critically to human health and disease predisposition and pathophysiology.
Given the great importance of muramyl peptides in human health and disease, technologies in detecting, quantifying, neutralizing or sequestering these molecules are expected to have broad applications in disease prediction, diagnosis, and treatment. Such technologies can also be used to detect bacterial contamination of medical facilities, reagents, biological products, foods, and beverages.
Monoclonal antibodies raised against muramyl peptides are known in the art. One such antibody mAb2-4, which isotype is IgG2a, has been characterized in detail. However, so far, mAb2-4 has only been known to be used in immunostaining of tissues to detect the presence of peptidoglycan mainly in inflammatory tissues and macrophages. This antibody is not known to be used for detecting peptidoglycan or muramyl peptides in solution by assays such as enzyme-linked immunosorbent assay (ELISA). Additionally, mAb2-4 was found to have very low affinity to muramyl peptides. Inhibition assays using mAb2-4 showed that 50% inhibition of mAb2-4 binding to peptidoglycan by N-acetylmuramyl-L-alanyl-D-isoglutamine occurred only at concentrations higher than 1 mg/ml. Additionally, structural analysis of antigenic determinant showed that the mAb2-4 antibody recognizes the N-acetylmuramic acid linked to the dipeptide. As many bacterial species do not have the N-acetyl group in the muramic acid residue, mAb2-4 appears to be an antibody that has narrow specificity in recognizing bacterial species.
Another commonly used mouse monoclonal antibody for peptidoglycan is mAb2E9, which was developed by immunizing mice with partially purified peptidoglycan-polysaccharide complexes isolated from the feces of a healthy human. The affinity of this antibody to N-acetylmuramyl-L-alanyl-D-isoglutamine was found to be even lower than mAb2-4, and the antigenic determinant has not been defined. mAb2E9 has been used in immunostaining of tissues but never in ELISA.
Other monoclonal antibodies developed by immunizing mice with peptidoglycan isolated from Streptococcus mutans have also been described. Although these antibodies could recognize peptidoglycan prepared from multiple bacterial species including both Grain-positive and Gram-negative ones, the binding of such antibodies are known to be non-specific as they cannot be inhibited by N-acetylmuramyl-L-alanyl-D-isoglutamine and the antigenic determinant is not known.
In view of the low affinity, poorly defined antigenic determinant and narrow specificity of the currently available mouse monoclonal antibodies, there is a need to provide an alternative antibody that recognizes muramyl peptide.